Numerous ubiquitin ligases and potential substrates are present at multiple locations within the cell, yet ubiquitination occurs only at specific organelles. While the subcellular location where a substrate is ubiquitinated has functional consequences for the fate of the substrate, the mechanisms that specify this location are incompletely understood. In neurons, ubiquitination regulates synapse assembly and function by a host of selectively targeted pre- and post-synaptic ubiquitin ligases. The mechanisms that target these ligases to their subcellular locations are poorly understood and constitute the focus of this proposal. Our interest in these mechanisms stems from the role of the ubiquitin machinery in sporadic and familial forms of neurodegenerative diseases, such as Parkinson's disease. Exciting new data in our laboratory demonstrate that a member of the HECT (homologous to E6-AP carboxy terminus) superfamily of ubiquitin ligases, Nedd4, interacts with the adaptor protein complex-3 (AP-3). While Nedd4 acts at the late endosome, the mechanisms for targeting it to this compartment are unknown. Since the AP-3 complex acts as a vesiculation scaffold that brings together multiple proteins at endocytic organelles, we postulate that Nedd4 is recruited to endocytic compartments by interaction with AP-3. Our overall hypothesis is that HECT family ubiquitin ligases recognize specific membranous organelles by binding to organelle-restricted docking factors, such as AP-3. We will test our hypothesis through the following specific aims: (1) to determine if the AP-3 complex plays a role in subcellular localization of the Nedd4 ubiquitin ligase and (2) to determine if AP-3 regulates Nedd-4-dependent substrate recognition and post-translational modification. We will use subcellular fractionation and quantitative high-resolution immunofluorescence microscopy of fixed primary cultured neurons from wild-type and Nedd4- deficient mice and neuronal model cell lines in order to test our hypothesis. Our long-term goal is to define subcellular compartmentalization as a novel biological regulatory principle of ubiquitin machineries. Given the importance of the ubiquitin machinery to the normal and pathological synapse, our work will contribute new mechanistic understanding of the processes involved in the establishment and maintenance of synapses. PUBLIC HEALTH RELEVANCE: Relevance to human health and disease: The health of the brain depends on the maintenance of the connections between brain cells. Dysregulation of a protein modification machinery, the ubiquitination machinery, can lead to neurodegenerative disease such as Parkinson's disease. Our work will contribute details of the regulation of this machinery in both health and neurodegenerative diseases.